Elucidation of the molecular mechanism of a well-defined membrane transport system will contribute to an understanding of how cells selectively take up small organic molecules and to an understanding of the molecular basis for cellular transport deficiencies. Such a system catalyzes the transport of sugar across the cytoplasmic membrane of Staphylococcus aureus, and their concomitant conversion to sugar- phosphates, in which form they are accumulated to high internal concentrations. The selective uptake and phosphorylation of lactose and its analogs is catalyzed by four proteins, which comprise the lactose phosphotransferase system (lac PTS). The function of each lac PTS protein in an in vitro sugar phosphorylation system has been determined and correlated with the pattern of sugar transport deficiencies of mutant cells lacking each of the proteins. In the critical steps of the transport/phosphorylation process, lactose is bound selectively, rapidly translocated across the membrane and phosphorylated. Cellular energy is coupled to some or all of these steps, and their molecular mechanisms can be described in terms of the sequence and nature of the interactions among lactose, EII (lactose-specific cytoplasmic protein). These interactions will be investigated by binding studies with EII membrane preparations, homogeneous FIII, and lactose analogs. Since FIII is composed of three identical subunits, the importance of multiple phosphorlyation of FIII and of its trimeric structure will be determined. Similar experiments will be performed using membrane from transport-deficient mutant cells.